过表达羧肽酶E对高糖培养的小鼠肾小球系膜细胞增殖和凋亡的影响
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江苏省卫生健康委员会课题(H201458);淮安市533英才工程科研项目资助计划(HAA201744)


Effects of overexpressed carboxypeptidase E on the proliferation and apoptosis of mice glomerular mesangial cells cultured in high glucose
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    摘要:

    目的:探究羧肽酶E(carboxypeptidase E,CPE)对高糖培养的小鼠肾小球系膜细胞(glomerular mesangial cell,GMC)增殖、周期及凋亡的影响。方法:PCR法扩增CPE基因片段,将其插入表达载体 pcDNA3.1质粒中以构建重组质粒pcDNA3.1-CPE。将质粒转染GMC细胞,通过蛋白质免疫印迹法检测CPE标签蛋白、增殖标志物细胞周期蛋白D1(CyclinD1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)以及凋亡标记物Cleaved-caspase-3的表达;采用平板克隆、Cell Counting Kit-8(CCK-8)法以及流式分析法检测过表达CPE对高糖培养的GMC细胞增殖、细胞周期以及凋亡的影响。结果:成功构建CPE过表达的GMC细胞。平板克隆以及CCK-8实验表明,过表达CPE可以抑制GMC细胞的增殖能力。流式细胞学检测表明,与对照组相比,CPE过表达明显促进了细胞凋亡;细胞周期中G1期细胞明显增加,S期细胞明显减少,差异有统计学意义(P < 0.01)。结论:过表达CPE可以抑制高糖培养的小鼠GMC细胞增殖并诱导细胞凋亡。

    Abstract:

    Objective:This study aims to investigate the effects of carboxypeptidase E(CPE) on the cell proliferation,cell cycle and apoptosis of mice glomerular mesangial cells(GMC)cultured in high glucose. Methods:The CPE gene fragment was amplified by PCR and inserted into the expression vector pcDNA3.1 plasmid to construct the recombinant plasmid pcDNA3.1-CPE. The plasmid was transfected into GMC,and the expression of CPE-tagged protein,proliferation marker CyclinD1,proliferating cell nuclear antigen(PCNA)and apoptosis marker Cleaved-caspase-3 were detected by Western blot;plate cloning,cell counting kit-8(CCK-8)and flow cytometry were used to detect the effect of overexpressed CPE on the proliferation,cell cycle and apoptosis of GMC cells cultured in high glucose. Results:The overexpression of CPE in GMC cells was successfully constructed. Plate cloning and CCK-8 showed that overexpression of CPE could inhibit the proliferation of GMC. Flow cytometry showed that the overexpression of CPE significantly promoted the cell apoptosis compared with the control group;the number of cells in G1 phase was significantly increased,while that in S phase was significantly decreased(P < 0.01). Conclusion:CPE can inhibit the proliferation of mice GMC cultured in high glucose and induce cell apoptosis.

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张恒璐,陈天宇,王苏雨,李 婉,陆卫平.过表达羧肽酶E对高糖培养的小鼠肾小球系膜细胞增殖和凋亡的影响[J].南京医科大学学报(自然科学版),2020,(3):374-379

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  • 收稿日期:2019-09-21
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  • 在线发布日期: 2020-03-30
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